Bombesin is a 14 amino acid peptide which was first isolated from the skin of the frog Bombina bombina (Anastasi et al., Experientia, 1971, 27, 166) and has the sequence:                pGlu-Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2 (SEQ ID NO: 1)        
Gastrin releasing peptide (GRP) is a 27 amino acid peptide isolated from the porcine gut. The last ten amino acids at the C-terminus of gastrin releasing peptide correspond with one amino acid alteration (3) to the last ten amino acids of bombesin, viz:                H-Gly-Asn-His-Trp-Ala-Val-Gly-His-Leu-Met-NH2 (SEQ ID NO:2).        
It has been reported (J. H. Walsh and J. R. Reeve, Peptides 6, (3), 63–68, (1985)) that bombesin and bombesin-like peptides such as gastrin releasing peptide (GRP) are secreted by human small-cell lung cancer (SCI C) cells. It has been postulated (P. J. Woll and E. Rozengurt, PNAS 85, 1859–1863, (1988)) that gastrin releasing factor antagonists would bind competitively to bombesin receptors in animals and would therefore be of use in the treatment of SCLC and/or in the control of clinical symptoms associated with this disease and due to hypersecretion of this peptide hormone. Analogues of bombesin/GRP have been shown to inhibit the binding of gastrin releasing peptide to a SCLC cell line and to inhibit the growth of SCLC cells in-vitro and in-vivo (S. Mahmoud et al., Cancel Research, 1991, 51, 1798; Moody T W et al., Life Sci. 1995, 56, 521; Moody T W et al., Peptides, 1996, 17, 1337). After Bombesin/GRP cell receptors were established on SCLC cells, receptors were also found to be present on human prostate cells. Relic H et al., (Prostate, 1994, 25: 29–38) showed that the PC-3 and DU-145 human prostate cancer cell lines possess specific high-affinity receptors for bombesin/GRP and are suitable models for the evaluation of anti-neoplastic activity of new bombesin/GRP antagonists in the treatment of androgen-dependent prostate cancer. Bombesin also increased the penetration of the two human prostatic carcinoma cell lines, the relatively indolent LNCaP cells and the aggressively growing and invasive PC-3 cells, in an in vitro invasion of reconstituted basement membrane (Matrigel) (Hoosein N M et al., J Urol, 149(5): 1209–1213). High-affinity binding sites for GRP were found on human colorectal cancer tissue (Preston, SR. et al, Br. J. Can., 1995, 71, 1087), suggesting that bombesin-like peptides may have a role in the pathogenesis of colorectal cancer, and bombesin receptor antagonists may be of value in the treatment of receptor-positive tumours. Inhibitory effects of bombesin/GRP antagonist RC-3095 and somatostatin analogue RC-160 were also seen on growth of HT-29 human colon cancer xenografts in nude mice (Radulovic S et al., Acta Oncol, 1994, 33(6): 693–701).
Studies with the anti-bombesin/GRP antibodies lead to the hypothesis that it may be possible to disrupt the autocrine growth cycle of bombesin/GRP using designed peptide receptor antagonists. Since then several types of Bombesin antagonists have been reported. These antagonists have been defined by type and position of the substitutions of the natural sequence. Early receptor antagonists suffered from low potency, lack of specificity, and toxicity, which presented serious problems with their scientific and therapeutic use.
More recent work has concentrated on modification of the carboxy terminal (C-terminal) region of these peptides to interrupt the receptor interaction utilizing a variety of different types of C-terminal modified analogs. These have included incorporation of D-amino acids, non-peptide bonds for example (psi.
CH2NH), amide, and ester modifications. These alterations gave rise to certain peptides having, improved characteristics (Staley J et al., Peptides, 1991, 12(1): 145–9; Coy D H et al., J Natl Cancer Inst Monogr. 13:992, 13: 133–9). Other patents that describes bombesin and related analogs are:                U.S. Pat. No. 5,834,433 (1998)        U.S. Pat. No. 5,723,578 (1998)        U.S. Pat. No. 5,620,959 (1997)        U.S. Pat. No. 5,620,959 (1997)        U.S. Pat. No. 5,428,019 (1999)        U.S. Pat. No. 5,399,094 (1994)        U.S. Pat. No. 5,084,555 (1992)        
A Bombesin/GRP antagonist (RC-3940-II) was found to inhibit the proliferation of SW-1990 human pancreatic adenocarcinoma cells in vivo and in vitro (Qin, Y. et al., 1995, Int. J. Cancer, 63, 257). Similar effect was seen with bombesin/GRP antagonist RC-3095 on the growth of CFPAC-1 human pancreatic cancer cells transplanted to nude mice or cultured in vitro (Qin Y et al., Can Res, 1994, 54(4): 1035–41).
As reported earlier, the autocrine growth cycle of bombesin/GRP in SCLC can be disrupted by bombesin/GRP antagonists such as [Psi 13,14] bombesin. Several bombesin analogues were solid phase synthesized and incubated with intact SCLC cells at 37° C. in RPMI medium in a time course fashion (0–1080 minutes) to determine enzymatic stability. The proteolytic stability of the compounds was determined by subsequent HPLC analysis. [Psi 13, 14] Bombesin was found to be very stable to metabolic enzymes (TI/2=646 min.) and inhibited SCLC xenograft formation in vivo in a dose-dependent manner (Davis T P et al., Peptides, 1992, 13(2): 401–7).
Female athymic nude mice bearing xenografts of the MCF-7 MIII human breast cancer cell line were treated for 7 weeks with bombesin/GRP antagonist (DTpi6, Leu3, psi[CH2NH]—Leu14) bombesin (6–14)(RC-3095) injected administered biweekly in the form of microgranules releasing 45 μg/day. After 2 weeks of treatment, a significant inhibition of tumor volume was observed in the groups treated with RC-3095 alone or in combination with SB-75 (Yano T et al., Cancer, 1994, 73(4): 1229–38).
Pinski J. et al., (Int. J. Cancer, 1994, 57(4): 574–580), demonstrated for the first time that the growth of gastrin-responsive human gastric carcinoma MKN45 cell line xenografts in nude mice could be inhibited not only by somatostatin analogues, but also by administration of modern bombesin/GRP antagonists, such as RC-3095, or a combination of these. RC-3095 also effectively inhibited tumor growth in nude mice bearing xenografts of the human gastric cancer cell line Hs746T (Qin Y et al., J Cancer Res Clin Oncol, 1994,120(9):519–528).
This invention describes the preparation and use of peptide analogs of bombesin/GRP using constrained amino acids and their use for cancer therapy, alone, or in combination or as an adjunct to or with other chemotherapeutic agents and compounds.
The design of conformationally constrained bioactive peptide derivatives has been one of the widely used approaches for the development of peptide-based therapeutic agents. Non-standard amino acids with strong conformational preferences may be used to direct the course of polypeptide chain folding, by imposing local stereochemical constraints, in de novo approaches to peptide design. The conformational characteristics of α,α-dialkylated amino acids have been well studied. The incorporation of these amino acids restricts the rotation of φ, ψ angles, within the molecule, thereby stabilizing a desired peptide conformation. The prototypic member of α,α-dialkylated aminoacids, α-aminoisobutyric acid (Aib) or α,α-dimethylglycine has been shown to induce (β-turn or helical conformation when incorporated in a peptide sequence (Prasad and Balaram, (1984); CRC Crit. Rev. Biochem. 16, 307–347; Karle and Balaram (1990) Biochemistry 29, 6747–6756). The conformational properties of the higher homologs of α,α-dialkylated amino acids such as diethylglycine (Deg), di-n-propylglycine (Dpg) and di-n-butylglycine (Dbg) as well as the cyclic side chain analogs of α,α-dialkylated amino acids such as 1-aminocyclopentane carboxylic acid (Ac5c). 1-aminocyclohexane carboxylic acid (Ac6c), 1-aminocycloheptane carboxylic acid (Ac7c) and 1-aminocyclooctane carboxylic acid (Ac8c) have also been shown to induce folded conformation (Prasad et al., (1995), Biopolymers 35, 11–20; Karle et al., (1995); J. Amer. Chem. Soc. 117, 9632–9637). α,α-dialkylated amino acids have been used in the design of highly potent chemotactic peptide analogs (Prasad et al., (1996) Int. J. Peptidic Proteins RCS. 48, 312–318).
The present invention exploits the conformational properties of α,α-dialkylated amino acids for the design of biologically active peptide derivatives, taking bombesin as the model system under consideration. Furthermore, it has been shown that lipophilization of bioactive peptides improves their stability, bioavailability and the ability to permeate biomembranes (Dasgupta, P et al; 1999, Pharmaceutical Res. 16, 1047–1053; Gozes, 1, et al 1996, Proc. Natl. Acad. Sci. USA, 93, 427–432). In the present invention, we have also synthesized peptide derivatives having N-terminal alkanoyl groups from C2–C16 carbon atoms, which retain anticancer activity.
The present invention exploits the conformational properties of α,α-dialkylated amino acids for the design of biologically active peptide derivatives, taking bombesin as the model system under consideration. Furthermore, it has been shown that lipophilization of bioactive peptides improves their stability, bioavailability and the ability to permeate biomembranes (Dasgupta, P et al; 1999, Pharmaceutical Res. 16, 1047–1053; Goes, L, et al., 1996, Proc. Natl. Acad. Sci. USA, 93, 427–432).
Throughout the specification and claims the amino acid residues are designated by their standard abbreviations. Amino acids denote L-configuration unless otherwise indicated by D or DL appearing before the symbol and separated from it by a hypen. Throughout the specification and claims, the following abbreviations are used with the following meanings:                BOP: Benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphonium hexfluorophosphate        PyBOP: Benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexofluorophospate        TBTU: 2-(1H-Benzotriazole-lyl)-1,1,3,3-tetramethyluronium tetrafluroborate        HBTU: O-Benzotriazole-N,N,N′,N′-tetramethyl-uronium-hexofluoro-phosphate        HOBt: 1-Hydroxy Benzotriazole        DCC: Dicyclohexyl carbodiimide        DIPCDI: Diisopropyl carbodiimide        DHEA: Diisopropyl ethylamine        DMF: Dimethyl formamide        DCM: Dichloromethane        NMP: N-Methyl-2-pyrrolidinone        TFA: trifluoroacetic acid        